18-20 member bi-polycyclic compounds

ABSTRACT

The invention relates to 18-20 member bi-polycyclic compounds, methods of making these compounds, and methods of using them in treating hyperproliferative disorders (e.g., cancer) and non-malignant tumors; promoting muscle formation; inhibiting muscle degeneration or the loss of muscle mass or muscle function; and myofibers ex vivo.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/316,029, filed Dec. 2, 2016, which is a U.S. national phaseapplication of International Application No. PCT/US2015/034303, filedJun. 4, 2015, which claims the benefit of the filing date of U.S.Provisional Application No. 62/007,673, filed Jun. 4, 2014, and U.S.Provisional Application No. 62/007,686, filed Jun. 4, 2014, thedisclosures of which are hereby incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The invention relates to 18-20 member bi-polycyclic compounds andmethods of making and using these compounds to promote muscle formation,inhibit muscle degeneration, and treat hyperproliferative disorders(e.g., cancer).

BACKGROUND OF THE INVENTION

The disappearance of muscle mass associated with several pathologicalconditions is not effectively addressed by palliative or pharmacologicaltherapies. Therefore there is a need to develop therapeutics that delayor prevent the mechanisms of muscle atrophy and can therefore inhibit orhelp to prevent muscle degeneration and support muscle function inpatients.

The following references may be of interest: Vannini et al., EMBO Rep. 8(9):879-84, 2007; Shibata et al., Recent Prog. Horm. Res. 52:141-64,1997; Gronemeyer and Miturski, Cell Mol. Biol. Lett. 6:3-52, 2001; Oehmeet al., Clin. Cancer Res. 15(1):91-9, 2009; Pori, Eur. J. Med. Chem.70:857-63, 2013; Evans et al., J. Clin. Nutr. 91(suppl):1123S-7S, 2010;Abmayr and Pavlath, Development 139:641-656, 2012; Macpherson et al., J.Cell. Biochem. 112(8):2149-59, 2010; Moresi et al. Cell 143:35-45, 2010;Nebbioso et al., EMBO Rep. 10(7):776-82, 2009; Glenisson et al.,Biochim. Biophys. Acta. 1773:1572-82, 2007 and WO/2011/097712.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a compound of Formula I:A-W—Z (I) or a pharmaceutically acceptable salt thereof, wherein A is

W is a heterocyclylene, arylene, heteroarylene, alkenylenearylene,arylenealkenylene alkenyleneheteroarylene, or heteroarylenealkenylene;and

Z is a hydrogen bond donor, with the proviso that the compound is not

where R is —OH, —OCH₃ and —NHOH; and R′ is —OH or —OCH₃.

In another aspect, the invention features methods of treating a subjectwho has cancer or a non-malignant tumor by administering to the subjecta therapeutically effective amount of a compound described above or apharmaceutically acceptable composition containing such a compound or amixture thereof.

The invention also relates, in part, to methods of inhibiting loss ofmuscle mass or muscle function in a subject, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a compound of Formula I:A-W—Z   (I)

or a pharmaceutically acceptable salt thereof, wherein

A is

W is a heterocyclylene, arylene, heteroarylene, alkenylenearylene,arylenealkenylene or alkenyleneheteroarylene, heteroarylenealkenylene;and

Z is a hydrogen bond donor.

In another aspect, the invention features methods for treating myofibersex vivo. These methods can include the steps of providing an ex vivopreparation of myofibers, optionally comprising a natural or syntheticbiological matrix; and contacting the preparation with an amount of acompound of Formula I: A-W—Z (I) or a pharmaceutically acceptable saltthereof, that is sufficient to promote muscle mass or muscle function.Within Formula I,

A is

W is a heterocyclylene, arylene, heteroarylene, alkenylenearylene,arylenealkenylene alkenyleneheteroarylene, or heteroarylenealkenylene;and Z is a hydrogen bond donor.

In another aspect, the invention features methods of making a compoundas described herein, for example, as illustrated in the syntheticschemes shown below.

In other aspects, the invention features the use of one or more of thecompounds described herein in the preparation of a medicament or in thepreparation of a medicament for the treatment of cancer to forinhibiting the loss of muscle mass or muscle function.

Where elements are listed, it is to be understood that any one or moreof the listed elements can be excluded from the compound, composition,or method. For example, the inventors have specified that W can be aheterocyclylene, arylene, heteroarylene, alkenylenearylene,arylenealkenylene alkenyleneheteroarylene, or heteroarylenealkenylene.Therefore, W can be any of these elements except heterocyclylene, forexample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 exemplifies various compounds of embodiments of the invention,Compound 13, Compound 5, Compound 17, Compound 8, Compound B, SAHA,Compound 18, Compound 19, Compound 20 and Compound 21.

FIG. 2 shows a proton NMR spectra for compound 5.

FIG. 3 shows a proton NMR spectra for compound 8.

FIG. 4 shows a proton NMR spectra for compound 13.

FIG. 5A and FIG. 5B are graphs demonstrating dose-dependent inhibitionof recombinant human HDAC-1 and HDAC-6 using compounds disclosed herein.

FIG. 6A and FIG. 6B are graphs showing the induction of RAR-dependenttranscriptional activity and lack of induction of RXR-dependenttranscriptional activity in MCF7 cells.

FIG. 7A, FIG. 7B and FIG. 7C are graphs depicting the cytotoxic effectof compounds disclosed herein in high risk neuroblastoma (IMR-32 andBE-2C) and triple negative breast cancer (MDA-MB-231) cell lines.

FIG. 8 is a series of gels showing the effect of compound 13 onintracellular acetylation of p53 protein in IMR-32 neuroblastoma cells.

FIG. 9 shows a series of gels demonstrating the effect of certaincompound on intracellular acetylation of alpha-tubulin in BE-2Cneuroblastoma cells.

FIG. 10 is a series of photomicrographs showing immunohistochemicallystained C2Cl2 cells in culture and the effect of certain compounds onfiber apparition and maintenance in long term culture.

FIG. 11 shows how the certain compounds of the invention affect fusionof differentiated mononuclear C2Cl2 myoblasts in long term cultures.

FIG. 12A and FIG. 12B are bar graphs showing the effect of compound 17on the distribution of the length and diameter of myotubes in long termC2Cl2 culture.

FIG. 13 shows the effect of certain compounds on myotube degeneration indifferentiated C2Cl2 cells.

FIG. 14A and FIG. 14B are bar graphs showing the effect of compounds 13and 17 on myofiber gene expression in C2Cl2 cells.

FIG. 15 is a graph comparing the pharmacokinetic profile for compounds13 and 17.

DETAILED DESCRIPTION

Compounds: The invention relates to compounds of Formula I: A-W—Z (I) ora pharmaceutically acceptable salt thereof, wherein A is

W is a heterocyclylene, arylene, heteroarylene, alkenylenearylene,arylenealkenylene alkenyleneheteroarylene, or heteroarylenealkenylene;and

Z is a hydrogen bond donor, with the proviso that the compound is not acompound conforming to

wherein Z is a hydrogen bond donor (e.g., wherein Z is —OH, —O(C₁₋₆alkyl), —NH₂, and —NHOH). For example, the invention relates tocompounds of Formula I with the proviso that the compound is not:

where R is —OH, —OCH₃ and —NHOH; and R′ is —H or —OCH₃.

In some embodiments, W is an indolinylene linked to A at any one ofpositions 2, 3, 4, 5, 6 or 7 of the indolinylene; a quinolinene linkedto A at any one of positions 2, 3, 4, 5, 6, 7, or 8; or anisoquinolinene linked to A at any one of positions 1, 3, 4, 5, 6, 7, or8. In other embodiments, W is -propylene-phenylene-.

In regard to the indolinylene, quinolinene and isoquinolinene moieties,the following numbering schemes apply:

The compounds of the invention are not limited to the indolinylene,quinolinene and isoquinolinene moieties for W. The nitrogen atom inthese ring systems may have an oxygen or sulfur atom instead ofnitrogen.

In some embodiments, W is

In some embodiments, Z is —C(O)NR¹R² or —C(O)OR³, where R¹ and R² areeach independently hydrogen (H), hydroxyl (OH), C₁₋₆ alkyl, hydroxyC₁₋₆alkyl, aminoC₁₋₆ alkyl, or aminoaryl; and R³ is H or C₁₋₆ alkyl.

In some embodiments, Z is linked to the indolinylene, quinolinene, orisoquinolinene at any one of the positions of the indolinylene,quinolinene, or isoquinolinene that is not linked to A.

In some embodiments, W is an indolinylene linked to A at any one ofpositions 2, 3, 4, 5, 6 or 7 of the indolinylene; Z is —C(O)NR¹R² or—C(O)OR³, where R¹ and R² are each independently hydrogen, hydroxyl,C₁₋₆ alkyl, hydroxyC₁₋₆ alkyl, aminoC₁₋₆ alkyl, or aminoaryl; and R³ isH or C₁₋₆ alkyl, and where Z is linked to the indolinylene at any one ofpositions 2, 3, 4, 5, 6 or 7 of the indolinylene not linked to A.

In other embodiments, W is a quinolinene linked to A at any one ofpositions 2, 3, 4, 5, 6, 7 or 8 of the quinolinene; Z is —C(O)NR¹R² or—C(O)OR³, where R¹ and R² are each independently hydrogen, hydroxyl,C₁₋₆ alkyl, hydroxyC₁₋₆ alkyl, aminoC₁₋₆ alkyl, or aminoaryl; and R³ isH or C₁₋₆ alkyl, and where Z is linked to the quinolinene at any one ofpositions 2, 3, 4, 5, 6, 7 or 8 of the quinolinene.

In certain other embodiments, W is a isoquinolinene linked to A at oneof positions 1, 3, 4, 5, 6, 7 or 8 of the isoquinolinene moiety; Z is—C(O)NR¹R² or —C(O)OR³, where R¹ and R² are each independently hydrogen,hydroxyl, C₁₋₆ alkyl, hydroxyC₁₋₆ alkyl, aminoC₁₋₆ alkyl, or aminoaryl;and R³ is H or C₁₋₆ alkyl, and where Z is linked to the quinoline ringat any one of positions 2, 3, 4, 5, 6, 7 or 8 of the isoquinolinene.

In one embodiment, Z is —C(O)NR¹R²; R¹ is H; and R² is OH.

In another embodiment, Z is —C(O)NR¹R²; R¹ is H; and R² is aminoaryl.

In yet another embodiment, Z is —C(O)OR³; and R³ is H.

In another embodiment, Z is —C(O)OR³; and R³ is C₁₋₆ alkyl.

The compound can be:

It is to be understood that certain features of the invention, whichare, for clarity, described in the context of separate embodiments, canbe combined in a single embodiment. Conversely, various features of theinvention which are, for brevity, described in the context of a singleembodiment, can also be provided separately or in any suitablesubcombination.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Accordingly, thecompositions of the invention (e.g., pharmaceutical compositions) caninclude a compound or compounds in the R-form, the S-form, or a racemicor non-racemic mixture thereof. Compounds that contain asymmetricallysubstituted carbon atoms can be isolated in optically active or racemicforms. Methods for preparing optically active forms from opticallyinactive starting materials are known in the art and include resolutionof racemic mixtures and stereoselective synthesis. Stable, geometricisomers are also contemplated in the present invention. Cis and transgeometric isomers of the compounds are described and may be isolated asa mixture of isomers or as separate isomers.

Resolution of racemic mixtures can be carried out by any of the numerousmethods known in the art. For example, compounds can be resolved byfractional recrystallization using a chiral resolving acid which is anoptically active, salt forming organic acid. Suitable resolving agentsfor fractional recrystallization methods are, for example, opticallyactive acids, such as the D and L forms of tartaric acid,diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malicacid, lactic acid or the various optically active camphorsulfonic acidssuch as 3-camphorsulfonic acid. Other resolving agents suitable forfractional crystallization methods include stereoisomerically pure formsof ct-methylbenzylamine (e.g., S and R forms, or diastereomerically pureforms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine,cyclohexylethylamine, 1,2-diaminocyclohexane, and the like. Racemicmixtures can also be resolved by elution on a column packed with anoptically active resolving agent (e.g., dinitrobenzoylphenylglycine).Suitable elution solvent compositions can be determined by one ofordinary skill in the art.

Compounds of the invention also include tautomeric forms, which resultfrom the swapping of a single bond with an adjacent double bond togetherwith the concomitant migration of a proton. Tautomeric forms includeprototropic tantomers which are isomeric protonation states having thesame empirical formula and total charge. Examples of prototropictautomers include ketone-enol pairs, amide-imidic acid pairs,lactam-lactim pairs, amide-imidic acid pairs, enamine-imine pairs, andannular forms where a proton can occupy two or more positions of aheterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H- and4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by an appropriate substitution.

Compounds of the invention can also include all isotopes of atomsoccurring in the intermediates or final compounds. Isotopes includethose atoms having the same atomic number but different mass numbers.For example, isotopes of hydrogen include tritium and deuterium.

As used herein, the term “alkyl” refers to a saturated hydrocarbon groupwhich is straight-chained or branched. Examples of alkyl groups includemethyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl(e.g., n-butyl, isobutyl, sec-butyl, t-butyl), pentyl (e.g., n-pentyl,isopentyl, sec-pentyl, neopentyl), and the like. An alkyl group cancontain from 1 to about 20 (e.g., from 2 to about 20, from 1 to about10, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1to about 3) carbon atoms. By “about,” we mean ±10% (e.g., about 20 gramsis 18-22 grams) or, where the referenced number of units is less than 10or not readily useful in divided tenths, we mean ±1 (e.g., about 6carbon atoms is 5-7 carbon atoms; about 20 carbon atoms is 19-21 carbonatoms).

The term “alkoxy,” when used alone or in combination with other groups,refers to a terminal oxy containing alkyl group, as defined above suchas methoxy, ethoxy, propoxy, isopropoxy and the like.

The terms “alkenyl” and “alkynyl,” when used alone or in combinationwith other groups, refer to mono- or polyunsaturated aliphatichydrocarbon radicals containing from two to 15 carbon atoms and at leastone double or triple bond, respectively. “Alkenyl” and “alkynyl” referto both branched and unbranched alkenyl and alkynyl groups,respectively. The alkenyl and alkynyl groups include straight chainedalkenyl or alkynyl groups containing from two to eight carbon atoms andbranched alkenyl or alkynyl groups containing from five to ten carbonatoms. The alkenyl and alkynyl groups also include alkenyl and alkynylgroups containing from two to six carbon atoms and branched alkenyl andalkynyl groups containing from 5 to eight carbon atoms. Examples ofalkenyl groups include ethenyl, 2-propenyl, 1-propenyl,2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl,3-pentenyl, allyl, 1,3-butadienyl, 1,3-dipentenyl, 1,4 dipentenyl,1-hexenyl, 1,3-hexenyl, 1,4-hexenyl, 1,3,5-trihexenyl, 2,4-dihexenyl,and the like.

Examples of alkynyl include ethynyl, 1-propynyl, 2-propynyl, 1 butynyl,2-butynyl, 2-methyl-1-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl,3-methyl-1-pentynyl, 2-methyl-1-pentynyl, 1-hexynyl, 2-hexynyl,3-hexynyl, and the like. The alkenyl and alkynyl groups contain at leastone double bond or one triple bond, respectively. In another embodiment,they each may contain up to 4 carbon-carbon multiple bonds, for example,1, 2, 3, or 4, double bonds or triple bonds, respectively. The doublebonds in the alkenyl groups may be conjugated, as in 1,3-butadienyl, ornon-conjugated, as in 1,4-di pentenyl.

As used herein, “aryl” refers to monocyclic or polycyclic (e.g., having2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example,phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and thelike. In some embodiments, aryl groups have from 6 to about 20 carbonatoms. The term “aryl” includes aromatic rings fused to non-aromaticrings, as long as one of the fused rings is an aromatic hydrocarbon.

The term “heteroaryl” refers to an aryl group in which at least one ofthe ring atoms is a heteroatom (i.e., oxygen, nitrogen, or sulfur).Heteroaryl groups include monocyclic and polycyclic (e.g., having 2, 3or 4 fused rings) systems. Examples of heteroaryl groups include withoutlimitation, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl,furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl,pyrryl, oxazolyl, benzodiazine, benzofuryl, benzothienyl, benzthiazolyl,isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl,1,2,4-thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl,benzimidazolyl, indolinyl, quinolinyl, isoquinolinyl purinyl, indolyl,and the like.

The term “heterocyclic” when used alone or in combination with othergroups refers to a 5- to 8-membered (e.g., 5- or 6-membered) monocyclicor 8- to 11-membered bicyclic heterocyclic radical that may be eithersaturated or unsaturated, aromatic or non-aromatic, and which may beoptionally benzo- or pyrido-fused if monocyclic, containing at least onering heteroatom. Each heterocycle consists of carbon atoms and from 1 to4 ring heteroatoms selected from nitrogen, oxygen and sulfur.

As used herein, “nitrogen” and “sulfur” include any oxidized form ofnitrogen and sulfur and the quaternized form of any basic nitrogen.

“Alkylene” refers to a linear or branched saturated divalent hydrocarbonradical, which may optionally be substituted as described herein. Incertain embodiments, the alkylene is a linear saturated divalenthydrocarbon radical that has 1 to 20 (C₁₋₂₀), 1 to 15 (C₁₋₁₅), 1 to 10(C₁₋₁₀), or 1 to 6 (C₁₋₆) carbon atoms, or branched saturated divalenthydrocarbon radical of 3 to 20 (C₃₋₂₀), 3 to 15 (C₃₋₁₅), 3 to 10(C₃₋₁₀), or 3 to 6 (C₃₋₆) carbon atoms. Linear C₁₋₆ and branched C₃₋₆alkylene groups are also referred as “lower alkylene.” Examples ofalkylene groups include, but are not limited to, methylene, ethylene,propylene (including all isomeric forms), n-propylene, isopropylene,butylene (including all isomeric forms), n-butylene, isobutylene,t-butylene, pentylene (including all isomeric forms), and hexylene(including all isomeric forms). For example, C₁₋₆ alkylene refers to alinear saturated divalent hydrocarbon radical of 1 to 6 carbon atoms ora branched saturated divalent hydrocarbon radical of 3 to 6 carbonatoms.

The term “alkenylene” refers to a linear or branched divalenthydrocarbon radical that contains one or more carbon-carbon doublebonds. The alkenylene may be optionally substituted as described herein.The term “alkenylene” also embraces radicals having “cis” and “trans”configurations, or alternatively, “E” and “Z” configurations. As usedherein, the term “alkenylene” encompasses both linear and branchedalkenylene, unless otherwise specified. For example, C₂₋₆ alkenylenerefers to a linear unsaturated divalent hydrocarbon radical of 2 to 6carbon atoms or a branched unsaturated divalent hydrocarbon radical of 3to 6 carbon atoms. In certain embodiments, the alkenylene is a lineardivalent hydrocarbon radical of 2 to 20 (C₂₋₂₀), 2 to 15 (C₂₋₁₅), 2 to10 (C₂₋₁₀), or 2 to 6 (C₂₋₆) carbon atoms, or a branched divalenthydrocarbon radical of 3 to 20 (C₃₋₂₀), 3 to 15 (C₃₋₁₅), 3 to 10(C₃₋₁₀), or 3 to 6 (C₃₋₆) carbon atoms. Examples of alkenylene groupsinclude, but are not limited to, ethenylene, allylene, propenylene,butenylene, and 4-methylbutenylene.

The term “cycloalkylene” refers to a cyclic saturated bridged and/ornon-bridged divalent hydrocarbon radical, which may be optionallysubstituted as described herein. In certain embodiments, thecycloalkylene has from 3 to 20 (C₃₋₂₀), from 3 to 15 (C₃₋₁₅), from 3 to10 (C₃₋₁₀), or from 3 to 7 (C₃₋₇) carbon atoms. Examples ofcycloalkylene groups include, but are not limited to, cyclopropylene(e.g., 1,1-cyclopropylene and 1,2-cyclopropylene), cyclobutylene (e.g.,1,1-cyclobutylene, 1,2-cyclobutylene, or 1,3-cyclobutylene),cyclopentylene (e.g., 1,1-cyclopentylene, 1,2-cyclopentylene, or1,3-cyclopentylene), cyclohexylene (e.g., 1,1-cyclohexylene,1,2-cyclohexylene, 1,3-cyclohexylene, or 1,4-cyclohexylene),cycloheptylene (e.g., 1,1-cycloheptylene, 1,2-cycloheptylene,1,3-cycloheptylene, or 1,4-cycloheptylene), decalinylene, andadamantylene.

The term “heterocyclylene” refers to a divalent non-aromatic ring systemand/or a multicyclic ring system that contains at least one non-aromaticring, wherein one or more of the non-aromatic ring atoms are heteroatomsindependently selected from O, S, or N, and the remaining ring atoms arecarbon atoms. In certain embodiments, the heterocyclylene group has from3 to 20 (e.g., 3-15, 3-10, 3-8, 4-7, or 5-6) ring atoms. In certainembodiments, the heterocyclylene is a monocyclic, bicyclic, tricyclic,or tetracyclic ring system, which may include a fused or bridged ringsystem, and in which the nitrogen or sulfur atoms may be optionallyoxidized, the nitrogen atoms may be optionally quaternized, and somerings may be partially or fully saturated, or aromatic. Theheterocyclylene may be attached to the main structure at any heteroatomor carbon atom which results in the creation of a stable compound.Examples of such heterocyclene groups include, but are not limited to,azepinylene, benzodioxanylene, benzodioxolylene, benzofuranonylene,benzopyranonylene, benzopyranylene, benzotetrahydrofuranylene,benzotetrahydrothienylene, benzothiopyranylene, benzoxazinylene,β-carbolinylene, chromanylene, chromonylene, cinnolinylene,coumarinylene, decahydroisoquinolinylene, dihydrobenzisothiazinylene,dihydrobenzisoxazinylene, dihydrofurylene, dihydroisoindolylene,dihydropyranylene, dihydropyrazolylene, dihydropyrazinylene,dihydropyridinylene, dihydropyrimidinylene, dihydropyrrolylene,dioxolanylene, 1,4-dithianylene, furanonylene, imidazolidinylene,imidazolinylene, indolinylene, isobenzotetrahydrofuranylene,isobenzotetrahydrothienylene, isochromanylene, isocoumarinylene,isoindolinylene, isothiazolidinylene, isoxazolidinylene, morpholinylene,octahydroindolylene, octahydroisoindolylene, oxazolidinonylene,oxazolidinylene, oxiranylene, piperazinylene, piperidinylene,4-piperidonylene, pyrazolidinylene, pyrazolinylene, pyrrolidinylene,pyrrolinylene, quinuclidinylene, tetrahydrofurylene,tetrahydroisoquinolinylene, tetrahydropyranylene, tetrahydrothienylene,thiamorpholinylene, thiazolidinylene, tetrahydroquinolinylene, and1,3,5-trithianylene.

The terms, arylene, heteroarylene, alkenylenearylene, arylenealkenylenealkenyleneheteroarylene, or heteroarylenealkenylene all refer todivalent forms of aryl, heteroaryl, alkenylaryl, arylalkenyl,alkenylheteroaryl and heteroarylalkenyl radicals.

As used herein, a “hydrogen bond donor” comprises a hydrogen atomattached to an electronegative atom (e.g., fluorine, oxygen, sulfur ornitrogen). In some embodiments, the hydrogen bond donor is —OH, —O(C₁₋₆alkyl), —NH₂, or —NHOH.

The term “compound,” as used herein is meant to include allstereoisomers, geometric isomers, tantomers, and isotopes of thestructures depicted, including structures conforming to a genericformula set out herein.

All compounds, and all pharmaceutically acceptable salts thereof, can beeither found together with other substances such as water and solvents(e.g., hydrates and solvates) or can be substantially isolated. Acompound of the invention or a salt thereof is “substantially isolated”when at least partially or substantially separated from the environmentin which it was formed or detected. Partial separation can include, forexample, a composition enriched to any extent in a compound of theinvention. Substantial separation can include compositions containing atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 97%, or atleast about 99% by weight of a compound or compounds of the invention,or a salt or salts thereof. Methods for isolating compounds and theirsalts are routine in the art.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The expressions “ambient temperature” and “room temperature,” as usedherein, are understood in the art, and refer generally to a temperature,e.g. a reaction temperature, that is about the temperature of the roomin which the reaction is carried out, for example, a temperature fromabout 20° C. to about 30° C.

The present invention also includes pharmaceutically acceptable salts ofthe compounds described herein. As used herein, “pharmaceuticallyacceptable salts” are derivatives of the disclosed compounds wherein theparent compound is modified by converting an existing acid or basemoiety to its salt form. Examples of pharmaceutically acceptable saltsinclude, but are not limited to, mineral or organic acid salts of basicresidues such as amines; alkali or organic salts of acidic residues suchas carboxylic acids; and the like. The pharmaceutically acceptable saltsof the present invention include the conventional non-toxic salts of theparent compound formed, for example, from non-toxic inorganic or organicacids. The pharmaceutically acceptable salts of the present inventioncan be synthesized from the parent compound which contains a basic oracidic moiety by conventional chemical methods. Generally, such saltscan be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, non-aqueous media like ether, ethyl acetate, alcohols (e.g.,methanol, ethanol, iso-propanol, or butanol) or acetonitrile (ACN) arepreferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), eachof which is incorporated herein by reference in its entirety.

Synthesis: The reactions for preparing compounds of the invention can becarried out in suitable solvents which can be readily selected by one ofskill in the art of organic synthesis. Suitable solvents can besubstantially non-reactive with the starting materials (reactants), theintermediates, or products at the temperatures at which the reactionsare carried out, e.g., temperatures which can range from the solvent'sfreezing temperature to the solvent's boiling temperature. A givenreaction can be carried out in one solvent or a mixture of more than onesolvent. Depending on the particular reaction step, suitable solventsfor a particular reaction step can be selected by the skilled artisan.

Preparation of compounds of the invention can involve the protection anddeprotection of various chemical groups. The need for protection anddeprotection, and the selection of appropriate protecting groups, can bereadily determined by one skilled in the art. The chemistry ofprotecting groups can be found, for example, in T. W. Greene and P. G.M. Wuts, Protective Groups in Organic Synthesis, 3^(rd) Ed., Wiley &Sons, Inc., New York (1999), which is incorporated herein by referencein its entirety.

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., 1H or13C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), massspectrometry, or by chromatographic methods such as high performanceliquid chromatography (HPLC) or thin layer chromatography (TLC).

An exemplary method for preparing certain compounds of the invention isprovided in Scheme 1.

Compound 3 was produced by bromination of methyl indole-5-carboxylate,compound 2 (ACC Corporation). Compound 4 is then produced by reactingcompound 1 with compound 3. Compound 5 was prepared from thecorresponding methyl carboxylic ester, compound 4, by nucleophilicsubstitution with hydroxylamine in the presence of NaOH and MeOH.

Scheme 2 shows the synthetic scheme for producing compound 8, which wasderived from compound 7 by replacement of methyl ester by hydroxamate inthe presence of NaOH and MeOH/CH₂Cl₂. Compound 7 was the product of aSuzuki coupling between compound 1 and methyl6-bromoquinoline-2-carboxylate, compound 6.

Further, Scheme 3 shows the synthetic scheme for preparing compound 13,which is derived from compound 12 by replacement of the methyl ester byhydroxamate in the presence of NaOH and MeOH/CH₂Cl₂. Compound 12 wasprepared by performing concomitant intramolecular Pd(OAc)₂-catalysedBuchwald-Hartwig coupling of vinyl bromide and amine in compound 11followed by intermolecular Suzuki cross coupling of the resulting2-bromoindole with boronic acid,(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) (compound 1).Compound 11 was synthesized from compound 10 by platinium/carboncatalytic hydrogenation of the nitro group. 1,1-Dibromoalkene compound10 was synthesized by Corey-Fuchs reaction with aldehyde 9.

Scheme 4 shows a synthetic method for preparing compound 17.

Methods and Use of the present compounds in the preparation of amedicament: In another aspect, the invention features methods of use ormethods of treatment for a patient who is suffering from ahyperproliferative disorder (e.g., a cancer, including blood-bornecancers and cancers that manifest as solid tumors). The “use” or methodsof use can include the preparation of a medicament or the preparation ofa medicament for treating a hyperproliferative disorder, non-malignanttumor, or muscle-related condition as described herein. Themedicament(s) can include a compound or compounds as described herein(e.g., for the treatment of a hyperproliferative disorder), and themethods of treating a patient can include a step of administering to thepatient a therapeutically effective amount of a compound or compounds asdescribed herein. For ease of reading, we do not repeat the phrase “or apharmaceutically acceptable salt thereof” at every opportunity. It is tobe understood that where a compound of the invention can be used, apharmaceutically acceptable salt thereof can also be used. In any methodof treatment, the method can include a step of identifying a patient inneed.

In one aspect, the invention relates to methods of treating a subjectwho has cancer or a non-malignant tumor, the method comprisingadministering to the subject a therapeutically effective amount of acompound described herein.

In various embodiments, the use can be directed to, or the methods oftreating can be applied to, a subject who has been diagnosed with lungcancer (e.g., a non-small cell lung cancer), a colon cancer, a melanoma,a breast cancer, a renal cancer, an ovarian cancer, a prostate cancer, acancer of the nervous system (affecting the brain or spinal cord, suchas a glioma), a neuroendocrine tumor (e.g., a neuroblastoma), or a bloodcancer (e.g., a leukemia or lymphoma).

In one embodiment, the present invention features methods of treatingpheochromocytoma in a subject comprising administering to the subject atherapeutically effective amount of a compound of Formula I or apharmaceutically acceptable salt thereof.

In another embodiment, the invention features methods of treatingneuroblastoma in a subject comprising administering to the subject atherapeutically effective amount of compound of Formula I or apharmaceutically acceptable salt thereof.

As noted, the present invention further provides a compound of FormulaI, or a pharmaceutically acceptable salt thereof for use in thepreparation of a medicament or for the production of a medicament foruse in therapy, including therapy for any condition or type of conditiondescribed herein.

Although the invention is not limited to compounds that exert abeneficial effect through any particular mechanism of action, ourstudies indicate that the compounds of the invention can inhibit tumorgrowth or survival. Compounds of the invention can also be an effectivetreatment to reduce metastatic growth of tumors (metastasis).

The present invention further provides methods for treating a diseasecaused by or associated with the presence of a tumor in a mammaliansubject, including identifying a subject in which reduction of tumormass is desirable, and administering to the subject in need of suchtreatment a therapeutically effective amount or dose of a compound ofthe present invention or a pharmaceutical composition thereof. Thesubject can be a mammal (e.g., a human or veterinary subject). Thecompound(s) and composition(s) described herein can be administeredorally or parenterally. The disease can be a solid extracranial tumorsuch as, but not limited to, neuroblatoma. The tumor can also beassociated with a breast cancer or be an intracranial tumor such, butnot limited to, a glioma. As noted, the disease can also be a bloodborne cancer such as, but not limited to, leukemia. In some embodiments,the disease is localized to a tissue or organ while in other instancesthe disease has spread to multiple organs or tissues (e.g., as inmetastasis).

As used herein, the term “contacting” refers to the bringing together ofindicated moieties (e.g., a compound or composition of the invention) inan in vitro system (e.g., a culture system including muscle cells orprecursors or progenitors thereof) or an in vivo system (e.g., withinthe body, either in the vicinity of or distant from a tumor) in a mannerthat produces a desired outcome (e.g., a treatment as described herein).

As noted, the present invention also relates to methods inhibiting theloss of muscle mass or muscle function in a subject, and such methodscan be carried out by administering to a subject in need thereof atherapeutically effective amount of a compound of Formula I: A-W—Z (I)or a pharmaceutically acceptable salt thereof, where

A is

W is a heterocyclylene, arylene, heteroarylene, alkenylenearylene,arylenealkenylene or alkenyleneheteroarylene, heteroarylenealkenylene;and Z is a hydrogen bond donor.

In certain aspects, the loss of muscle mass is associated with aninherited myopathy. in other aspects, the loss of muscle mass isassociated with muscular dystrophy, neuromyotonia, nemaline myopathy,multi/minicore myopathy, centronuclear myopathy, mitochondrial myopathy,inflammatory myopathy, metabolic myopathy. In yet another aspect, theloss of muscle mass is associated with intensive care unit-acquiredweakness (ICUAW), chronic obstructive pulmonary disease (COPD), heartfailure, traumatic injury or malignancy.

The invention also relates to methods for treating myofibers ex vivo.These methods can include the steps of providing an ex vivo preparationof myofibers, optionally comprising a natural or synthetic biologicalmatrix; and contacting the preparation with an amount of a compound ofFormula I: A-W—Z (I) or a pharmaceutically acceptable salt thereof,where the amount of the compound is sufficient to promote muscle mass ormuscle function and

A is

W is a heterocyclylene, arylene, heteroarylene, alkenylenearylene,arylenealkenylene alkenyleneheteroarylene, or heteroarylenealkenylene;and Z is a hydrogen bond donor.

As used herein, the terms “individual” or “patient,” or “subject” areused interchangeably (unless the context clearly indicates otherwise) torefer to any animal, including mammals, preferably mice, rats, otherrodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates,and most preferably humans.

As used herein, the phrase “therapeutically effective amount” refers tothe amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response that is being sought in a tissue,system, animal, individual or human by a researcher, veterinarian,medical doctor or other clinician.

As used herein, the term “treating” or “treatment” refers to one or moreof (1) preventing the disease; for example, preventing a disease,condition or disorder in an individual who may be predisposed to thedisease, condition or disorder but does not yet experience or displaythe pathology or symptomatology of the disease; (2) inhibiting thedisease; for example, inhibiting a disease, condition or disorder in anindividual who is experiencing or displaying the pathology orsymptomatology of the disease, condition or disorder (e.g., inhibitingthe progression of the disease); and (3) ameliorating the disease; forexample, ameliorating a disease, condition or disorder in an individualwho is experiencing or displaying the pathology or symptomatology of thedisease, condition or disorder (i.e., reversing the pathology and/orsymptomatology) such as decreasing the severity of disease.

Pharmaceutical Formulations and Dosage Forms: The invention providescompositions (e.g., pharmaceutical compositions) comprising a compoundof Formula I or a pharmaceutically acceptable salt thereof and at leastone carrier (e.g., a pharmaceutically acceptable carrier). Thus, theinvention relates to pharmaceutical compositions comprising a compoundas disclosed herein, and a pharmaceutically acceptable carrier. Thecompound can be present in an amount that confers a clinicallybeneficial result on a patient to whom the compound has beenadministered (i.e., a therapeutically effective amount).

When employed as pharmaceuticals, the compounds of the invention can beadministered in the form of pharmaceutical compositions. Thesecompositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by a variety of routes,depending upon whether local or systemic treatment is desired and uponthe area to be treated. For example, the compositions may beadministered orally or parenterally.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, the compound of the invention or apharmaceutically acceptable salt thereof, in combination with one ormore pharmaceutically acceptable carriers (excipients). In someembodiments, the composition is suitable for oral administration. Inmaking the compositions of the invention, the active ingredient istypically mixed with an excipient, diluted by an excipient or enclosedwithin such a carrier in the form of, for example, a capsule, sachet,paper, or other container. When the excipient serves as a diluent, itcan be a solid, semi-solid, or liquid material, which acts as a vehicle,carrier or medium for the active ingredient. Thus, the compositions canbe in the form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, soft and hardgelatin capsules, and sterile packaged powders. In preparing aformulation, the active compound can be milled to provide theappropriate particle size prior to combining with the other ingredients.If the active compound is substantially insoluble, it can be milled to aparticle size of less than 200 mesh. If the active compound issubstantially water soluble, the particle size can be adjusted bymilling to provide a substantially uniform distribution in theformulation, e.g. about 40 mesh.

The compounds of the invention may be milled using known millingprocedures such as wet milling to obtain a particle size appropriate fortablet formation and for other formulation types. Finely divided(nanoparticulate) preparations of the compounds of the invention can beprepared by processes known in the art, e.g., see InternationalApplication No. WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, alimentary oils andmethyl cellulose. The formulations can additionally include: lubricatingagents such as talc, magnesium stearate, and mineral oil; wettingagents; emulsifying and suspending agents; preserving agents such asmethyl- and propylhydroxy-benzoates; sweetening agents; and flavoringagents. The compositions of the invention can be formulated so as toprovide quick, sustained or delayed release of the active ingredientafter administration to the patient by employing procedures known in theart.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 to about 1000 mg (1 g), more usually about 100to about 500 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

In some embodiments, the compounds or compositions of the inventioncontain from about 5 to about 50 mg of the active ingredient. One havingordinary skill in the art will appreciate that this embodies compoundsor compositions containing about 5 to about 10, about 10to about 15,about 15 to about 20, about 20 to about 25, about 25 to about 30, about30 to about 35, about 35 to about 40, about 40 to about 45, or about 45to about 50 mg of the active ingredient.

In some embodiments, the compounds or compositions of the inventioncontain from about 50 to about 500 mg of the active ingredient. Onehaving ordinary skill in the art will appreciate that this embodiescompounds or compositions containing about 50 to about 100, about 100 toabout 150, about 150 to about 200, about 200 to about 250, about 250 toabout 300, about 350 to about 400, or about 450 to about 500 mg of theactive ingredient.

In some embodiments, the compounds or compositions of the inventioncontain from about 500 to about 1000 mg of the active ingredient. Onehaving ordinary skill in the art will appreciate that this embodiescompounds or compositions containing about 500 to about 550, about 550to about 600, about 600 to about 650, about 650 to about 700, about 700to about 750, about 750 to about 800, about 800 to about 850, about 850to about 900, about 900 to about 950, or about 950 to about 1000 mg ofthe active ingredient.

The active compound can be effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It willbe understood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, about 0.1 to about 1000 mg of the activeingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

In some embodiments, the compositions are administered by the oral routefor local effect. Solution, suspension, or powder compositions can beadministered orally from devices which deliver the formulation in anappropriate manner.

The amount of compound or composition administered to a patient willvary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications.Effective doses will depend on the disease condition being treated aswell as by the judgment of the attending clinician depending uponfactors such as the severity of the disease, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient can be in the form of apharmaceutical composition as described herein. These compositions canbe sterilized by conventional sterilization techniques, or may besterile filtered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain excipients, carriers, or stabilizers will result in theformation of pharmaceutical salts.

The therapeutic dosage of a compound of the present invention can varyaccording to, for example, the particular use for which the treatment ismade, the manner of administration of the compound, the health andcondition of the patient, and the judgment of the prescribing physician.

The proportion or concentration of a compound of the invention in apharmaceutical composition can vary depending upon a number of factorsincluding dosage, chemical characteristics (e.g., hydrophobicity), andthe route of administration. For example, the compounds of the inventioncan be provided in an aqueous physiological buffer solution containingabout 0.1 to about 10% w/v of the compound for parenteraladministration. Some typical dose ranges are from about 1 μg/kg to about1 g/kg of body weight per day.

EXAMPLES Example 1 Synthesis of Compound 21

Methyl 3-hydroxy-7-((trifluoromethylsulfonyl)oxy)-2-naphthoate (I1):3,7-dihydroxy-2-naphthoic acid (200 mg, 0.980 mmol, 1 eq) was dissolvedin methanol (5 mL) at room temperature. Concentrated HCl (7 drops,catalytic) was added, and the resulting mixture was stirred at refluxfor 16 hours. The reaction mixture was concentrated in vacuo andpartitioned between EtOAc (10 mL) and H₂O (10 mL). The aqueous layer wasfurther extracted with EtOAc (2×5 mL). The combined organic layer waswashed with brine, dried with anhydrous Na₂SO₄, filtered, andconcentrated in vacuo to give the corresponding methyl ester as a yellowsolid. No further purification was performed (the crude product was usedin the next step).

Characterization data for I1: δ_(H) (500 MHz, CDCl₃) 4.02 (s, 3H),7.12-7.17 (m, 2H), 7.27 (s, 1H), 7.60 (d, 1H, J 9 Hz), 10.28 (s, 1H).δ_(c) (500 MHz, CDCl₃) 52.6, 110.1, 111.8, 114.6, 121.6, 127.8, 128.2,130.4, 133.4, 151.8, 154.7, 170.3. HRMS (ESI) calculated for C₁₂H₉O₄(M−H⁺), 217.0501, found 217.0506.

I1 (214 mg, 0.975 mmol, 1 eq) was dissolved in dry DCM (5 ml). Thesolution was cooled to 0° C. and pyridine (158 μL, 1.96 mmol, 5 eq) andtriflic anhydride (165 μL, 0.975 mmol, 1 eq) were added using syringesin a dropwise manner. The reaction was allowed to warm up to roomtemperature and stirred for 2 hours. The reaction solution was dilutedwith Et₂O (5 mL) and quenched with excess HCl (aq., 3M). The aqueouslayer was further extracted with Et₂O (5 mL). The combined organic layerwas washed with saturated NaHCO₃ and brine, dried with anhydrous Na₂SO₄,filtered, and concentrated in vacuo to give a brown oil. The crudemixture was purified using flash chromatography (20% EtOAc in hexanes)to give I2 as an off-white solid (262 mg, 80% over 2 steps).

Characterization data for I2: δ_(H) (500 MHz, CDCl₃) 4.04 (s, 3H),7.34(s, 1H), 7.35 (dd, 1H, J2.5 Hz and 9.5 Hz), 7.69 (d, 1H, J 2.5 Hz), 7.72(dd, 1H, J 0.5 Hz and 9.5 Hz), 8.49 (d, 1H, J 0.5 Hz), 10.54 (s, 1H).δ_(c) (500 MHz, CDCl₃) 52.9, 112.1, 115.7, 120.3, 122.8, 126.3, 128.9,132.4, 136.6, 145.5, 157.3, 169.7. HRMS (ESI) calculated for C₁₃H₈F₃O₆S(M−H⁺), 358.9994, found 348.9999.

Methyl6-hydroxy-5′,5′,8′,8′-tetramethyl-5′,6′,7′,8′-tetrahydro-[2,2′-binaphthalene]-7-carboxylate(I4): I2 (13.0 mg, 0.0387 mmol, 1 eq) and I3 (27.0 mg, 0.116 mmol, 3 eq)were dissolved in DME (3 mL) at room temperature. PPh₃ (2.03 mg, 0.00773mmol, 20 mol %), KF dihydrate (11.0 mg, 0.116 mmol, 3 eq), and Pd₂bda₃(1.77 mg, 0.00193 mmol, 5 mol %) were added to the solution. Distilledwater (0.4 mL) was added to the resulting mixture and the reaction wasdegassed and purged with argon. The reaction mixture was stirred atreflux for 16 hours. After cooling, the reaction mixture was filteredthrough a layer of Celite and the filtrate partitioned between EtOAc (5mL) and H₂O (5 mL). The aqueous layer was further extracted with EtOAc(5 mL). The combined organic layer was washed with brine, dried withanhydrous Na₂SO₄, filtered and concentrated in vacuo. The crude mixturewas purified using flash chromatography (20% EtOAc in hexanes) to giveI4 (14.4 mg, 96%).

Characterization data for I4: δ_(H) (300 MHz, CDCl₃) 1.34 (s, 6H), 1.37(s, 6H), 1.74 (s, 2H), 4.04 (s, 3H), 7.33 (s, 1H), 7.43 (m, 2H), 7.61(s, 1H), 7.76 (m, 2H), 7.97 (s, 2H), 8.56 (s, 1H), 10.45 (s, 1H). δ_(c)(500 MHz, CDCl₃) 32.3, 32.5, 33.9, 34.1, 34.7, 34.9, 52.9, 125.2, 125.3,125.6, 125.9, 126.8, 127.4, 128.5, 129.1, 131.2, 131.9, 136.4, 137.7,141.7, 145.4, 145.7, 168.9.

Methyl5′,5′,8′,8′,-tetramethyl-6-((trifluoromethylsulfonyl)oxy)-5′,6′,7′,8′,-tetrahydo-[2,2′-binaphthalene]-7-carboxylate(I5): The phenol 14 (20.0 mg, 0.0257 mmol, 1 eq) was dissolved in dryDCM (2 ml). This solution was cooled to 0° C. and pyridine (20.8 μL,0.129 mmol, 5 eq) and triflic anhydride (13.0 μL, 0.0386 mmol, 1.5 eq)were added using a syringe. The reaction was allowed to warm up to roomtemperature and stirred for 2 hours. The reaction solution was dilutedwith Et₂O (5 mL) and quenched with excess HCl (aq., 3M). The aqueouslayer was further extracted with Et₂O (5 mL). The combined organic layerwas washed with saturated NaHCO₃ and brine, dried with anhydrous Na₂SO₄,filtered and concentrated in vacuo. The crude mixture was purified usingflash chromatography (20% EtOAc in hexanes) to give I5 (13.0 mg, 97%).

Characterization data for I5: δ_(H) (300 MHz, CDCl₃) 1.35 (s, 6H), 1.38(s, 6H), 1.75 (s, 4H), 4.03 (s, 3H), 7.47 (s, 2H), 7.63 (s, 1H), 7.76(s, 1H), 7.95 (s, 2H), 8.14 (s, 1H), 8.72 (s, 1H).

Methyl5′,5′,8′,8′-tetramethyl-5′,6′,7′,8′-tetrahydro-[2,2′-binaphthalene]-7-carboxylate(I6): The triflate I5 (29.4 mg, 0.0565 mmol, 1 eq) was dissolved in dryDMF (0.5 mL) at room temperature. DIPEA (29.5 μL, 0.169 mmol, 3 eq),Pd(PPh₃)₂Cl₂ (2 mg, 0.00283 mmol, 5 mol %), and HCOOH (43.0 μL, 0.113mmol, 2 eq) were added and the final solution was heated to 100° C. for6 hours. The cooled reaction mixture was filtered through a layer ofCelite and partitioned between EtOAc (5 mL) and H₂O (5 mL). The aqueouslayer was further extracted with another portion of EtOAc (5 mL). Thecombined organic layer was washed with brine, dried with anhydrousNa₂SO₄, filtered, and concentrated in vacuo. The crude mixture waspurified using flash chromatography (20% EtOAc in hexanes) to give I6(14.1 mg, 68%).

Characterization data for I6: δ_(H) (300 MHz, CDCl₃) 1.35 (s, 6H), 1.38(s, 6H), 1.75 (s, 4H), 4.00 (s, 3H), 7.43 (dd, 2H, J₁ 8.1 Hz and 8.1Hz), 7.65 (s, 1H), 7.84 (m, 3H), 8.04 (d, 1H, J 8.4 Hz), 8.12 (s, 1H),8.67 (s, 1H).

N-(benzyloxy)-5′,5′,8′,8′-tetramethyl-5′,6′,7′,8′-tetrahydro-[2,2′-binaphthalene]-7-carboxamide(I7): To a solution of I6 (14.1 mg, 0.0379 mmol, 1 eq) in dry THF (795μL) was added methanol (264 μL) and 1M KOH (aqueous, 264 μl, 7 eq). Thesolution was stirred at room temperature for 15 hours. The reactionmixture was acidified to around pH 3 with 3 M HCl, and extracted withEtOAc (3×5 mL). The combined organic layer was washed with brine, driedwith Na₂SO₄, filtered, and concentrated to dryness to give thecorresponding carboxylic acid. The crude acid (18.4 mg, 0.0513 mmol, 1eq) was then dissolved in dry DMF (2 mL), and OBnNH₂ hydrochloride salt(9.01 mg, 0.0564 mmol, 1.1 eq) and DIPEA (26.8 μL, 0.154 mmol, 3 eq)were added to the solution. HBTU (25.3 mg, 0.0667 mmol, 1.3 eq) wasadded. The resulting mixture was stirred at room temperature for 15hours. The crude reaction mixture was partitioned between EtOAc andwater. The organic layer was washed with brine, dried with Na₂SO₄,filtered, and concentrated in vacuo. The crude was purified by flashchromatography (6% EtOAc in toluene) to give I7 as a white solid (22.6mg, 95% over 2 steps).

Characterization data for I7: δ_(H) (300 MHz, CDCl₃) 1.33 (s, 6H), 1.36(s, 6H), 1.73 (s, 4H), 5.09 (s, 2H,), 7.28 (m, 1H), 7.32-7.38 (m, 5H),7.61 (d, 1H, J 1.2 Hz), 7.74-7.90 (m, 4H), 8.01 (s, 1H), 8.02 (s, 1H),8.29 (s, 1H).

N-hydroxy-5′,5′,8′,8′,-tetramethyl-5′,6′,7′,8′-tetrahydro-[2,2′-binaphthalene]-7-carboxamide(A): I7 (25.0 mg, 0.0539 mmol) was dissolved in minimal amounts ofEtOAc. A catalytic amount of Pd—C was added and the flask was purgedwith H₂ using a balloon. The reaction was further stirred at roomtemperature in presence of 1 atm of H₂ gas with TLC monitoring until thestarting material was completely consumed (about 20-30 min). Thereaction mixture was filtered through a layer of Celite and the filtrateconcentrated under reduced pressure to give an orange solid (15 mg, 85%before HPLC purification). The crude product was purified byreverse-phase HPLC (Agilent 1260 Infinity HPLC system equipped with anautosampler, a quaternary pump, a photodiode array detector, and anAgilent Zorbax SB-C18 analytical column, methanol/H₂O). The finalproduct A was obtained as a white crystalline solid.

Characterization data for A: δ_(H) (300 MHz, CDCl₃) 1.35 (s, 6H), 1.38(s, 6H), 1.75 (s, 4H), 7.43 (d, 1H, J 8.1 Hz), 7.47 (dd, 1H, J 2.1 Hzand 8.7 Hz), 7.64 (d, 1H, J 2.1 Hz), 7.83 (d, 2H, J 8.7 Hz), 7.92 (dd,2H, J 3.9 Hz and 8,1 Hz), 8.09 (m, 1H), 8.21 (s, 1H), 8.40 (s, 1H).δ_(c ()400 MHz, CDCl₃) 30.9, 31.2, 33.9, 34.3, 34.9, 35.2, 123.5, 124.4,124.6, 124.7, 125.3, 126.1, 127.0, 128.5, 129.3, 129.7, 131.9, 135.2,137.8, 140.7, 144.5, 145.1, 166.9. HRMS (ESI) calculated for C₂₅H₂₆NO₂(M−H⁺), 372.1969, found 372.1941.

Example 2 Synthesis of Compound B

1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphthalene (I8):2,5-dichloro-2,5-dimethylhexane (300 mg, 1.64 mmol, 1 eq) was dissolvedin dry benzene (25 mL). AlCl₃ (22.0 mg, 0.164 mmol, 0.1 eq) was added tothe solution, which was stirred at reflux for 16 hours. The reaction wasquenched with 3M HCl (5 mL) and extracted with hexanes (10 mL×3). Thecombined organic layer was washed with brine, dried with Na₂SO₄,filtered, and concentrated in vacuo. The crude was purified by flashchromatography (100% hexanes) to give I8 (309 mg, 91% yield) as acolourless oil.

Characterization data for I8: δ_(H) (500 MHz, CDCl₃) 1.33 (s, 12H), 1.74(s, 4H), 7.16-7.19 (m, 2H), 7.34-7.36 (m, 2H, J 2 Hz and 8.4 Hz).δ_(c)(500 MHz, CDCl₃) 31.9, 34.2, 35.1, 125.5, 126.5, 144.8.

1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethanone (I9):I8 (37.0 mg, 0.197 mmol, 1 eq) was dissolved in dry DCM (5 mL), to whichacetyl chloride (15.5 μL, 0.216 mmol, 1.1 eq) and AlCl₃ (26.2 mg, 0.216mmol, 1.1 eq) were added sequentially. The reaction mixture was refluxedfor two hours then cooled to room temperature and quenched with water.The crude mixture was extracted with EtOAc (3 mL×3). The combinedorganic layer was washed with NaHCO₃ (saturated, aqueous) and brine,dried with Na₂SO₄, filtered, and concentrated in vacuo. The crude waspurified by flash chromatography (10% EtOAc/hexanes) to give I9 (44.8mg, 98% yield).

Characterization data for I9: δ_(H) (500 MHz, CDCl₃) 1.28 (s, 6H), 1.30(s, 6H), 1.69 (s, 4H), 2.55 (s, 3H), 7.38 (d, 1H, J 8.0 Hz), 7.70 (dd,1H, J 1.9 Hz and 8.0 Hz), 7.92 (d, 1H, J 1.9 Hz). HRMS (ESI) calculatedfor C₁₆H₂₃O (M+H⁺), 231.1749, found 231.1767.

Ethyl4-(2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-1-en-1-yl)benzoate(I12): The Homer Wardsworth Emmons reagent I10 (316 mg, 1.10 mmol, 2 eq)was dissolved in dry THF (5 mL) and cooled to 0° C. NaHMDS (1M solutionin THF, 1.10 mL, 2 eq) was added dropwise to the phosphonate reagent andthe mixture stirred at low temperature for 30 minutes. 19 (127 mg, 0.552mmol, 1 eq) was dissolved in dry THF (5 mL) in a separate flask and wasslowly added to deprotonated HWE reagent through cannula. The mixturewas allowed to slowly warm up to room temperature and stirred untildisappearance of starting material. The reaction was quenched withsaturated NH₄Cl solution and extracted with EtOAc (5 mL×3). The combinedorganic layer was washed with NaHCO₃ (saturated, aqueous) and brine,dried with Na₂SO₄, filtered, and concentrated in vacuo. The crudeproduct was purified by flash chromatography (20% EtOAc/hexanes) to giveI11 as a E:Z mixture (1:4, 166 mg, 83% yield). I11 (166 mg, 0.458 mmol,1 eq) was dissolved in dry EtOH, to which an excess of freshly preparedNaOEt solution was added. The mixture was stirred at room temperaturefor two hours. Neutralizing workup and removal of organic solventyielded I12 as mostly E isomer.

Characterization data for I12 as an E isomer: δ_(H) (300 MHz, CDCl₃)1.32 (s, 6H), 1.35 (s, 6H), 1.39 (t, 3H, J 7.1 Hz), 1.72 (s, 4H), 2.30(d, 3H, J 1.2 Hz), 4.36 (q, 2H, J 7.1 Hz), 6.82 (s, 1H), 7.32 (m, 2H),7.42 (d, 1H, J 8.1 Hz), 7.47 (d, 1H, J 1.2 Hz), 8.04 (d, 1H, J 8.1 Hz).(E)-N-hydroxy-4-(2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-1-en-1-yl)benzamide(B):

I12 (49 mg, 0.135 mmol) was dissolved in THF (1 mL), and KOH (1M, 0.5mL) and MeOH (0.5 mL) were added. The reaction proceeded at roomtemperature for 5 hours until the starting material was not observableon TLC. The mixture was acidified with 3M HCl to pH 3 and extractedrepeatedly with EtOAc. The combined organic layer was washed with brine,dried with Na₂SO₄, filtered, and concentrated in vacuo. The crudecarboxylic acid was used without purification. The acid was subsequentlydissolved in dry DMF (2 mL). NH₂OTBS (23.3 mg, 0.158 mmol, 1.1 eq),DIPEA (75.0 μL, 0.430 mmol, 3 eq) were added with stirring at roomtemperature, followed by HBTU (71 mg, 0.186 mmol, 1.3 eq). The reactionmixture was stirred at room temperature for 16 hours. A quick extractionwith EtOAc was performed and the organic layer containing the O-TBSprotected hydroxamic acid was concentrated in vacuo and redissolved inDCM (3 mL). An excess of concentrated HF (˜20 eq) was added to the DCMsolution and the biphasic mixture was stirred vigorously for two hoursat room temperature. The reaction was neutralized with saturated NaHCO₃and extracted with additional DCM. The organic layer was washed withbrine, dried with Na₂SO₄, filtered, and concentrated in vacuo. The crudeproduct was purified by reverse-phase flash chromatography (20-95% MeOHin H₂O) to yield B as a slightly yellow oil (43 mg, 87%).

Characterization data for B: δ_(H) (500 MHz, CD₃OD) 1.31 (s, 6H), 1.34(s, 6H), 1.74 (s, 4H), 2.28 (d, 1H, J 1.5 Hz), 6.82 (s, 1H), 7.02 (dd,1H, J 2 Hz and 8.5 Hz), 7.31 (m, 2H), 7.45 (d, 2H, J 8.5 Hz), 7.77 (d,2H, J 8.5 Hz). δ_(c) (500 MHz, CD₃OD) 30.82, 30.84, 30.9, 33.7, 33.9,34.8, 35.0, 64.0, 123.1, 123.6, 125.3, 126.2, 126.6128.7, 128.9, 129.7,139.5, 140.7, 142.1, 144.0, 144.4, 166.6. HRMS (ESI) calculated forC₂₄H₂₈NO₂, [M−H⁺] 362.2123, found 362.2132.

Example 3 Synthesis of Compound 20

7-(benzyloxy)quinoline-3-carbonitrile (I13): I13 was prepared accordingto the procedure of Cai T. B., et al, Journal of Medicinal Chemistry,51:1849-1860, 2008.

Characterization data for I13: δ_(H) (500 MHz, CDCl₃) 5.28 (s, 2H),7.39-7.51 (m, 5H), 7.65 (dd, 1H, J 2.5 Hz and 9.0 Hz), 8.05 (d, 1H, J9.0 Hz), 8.14 (d, 1H, J 2.5 Hz), 8.63 (d, 1H, J 2.0 Hz), 9.15 (d, 1H, J2.0 Hz) δ_(c) (500 MHz, CDCl₃) 70.6, 116.3, 121.7, 122.3, 122.8, 123.1,123.3125.4, 127.7, 128.5, 129.5, 130.8, 141.2, 149.1, 151.4.

3-cyanoquinolin-7-yl trifluoromethanesulfonate (I14): I13 (27.0 mg,0.104 mmol, 1 eq) was dissolved in minimal EtOAc. A catalytic amount ofPd—C was added and the flask was purged with H₂ using a balloon. Thereaction was further stirred at room temperature in presence of 1 atm ofH₂ gas with TLC monitoring until the starting material was completelyconsumed (about three hours). The reaction mixture was filtered througha layer of Celite and the filtrate concentrated under reduced pressureto give an orange oil. The crude product was used in the next stepwithout purification. The crude hydroxyquinoline (17.7 mg, 0.104 mmol, 1eq) was dissolved in dry DCM (1 mL), pyridine (42.0 μL, 0.519 mmol, 5eq) and triflic anhydride (27.0 μL, 0.156 mmol, 1.5 eq) were added atroom temperature. The mixture was stirred for 2.5 hours at which pointthe reaction was quenched with water and partitioned between EtOAc andwater. The aqueous layer was extracted with additional EtOAc (5 mL×3)and the organic layers combined. The latter was washed with sodiumbicarbonate (saturated, aqueous) and brine, dried over anhydrous Na₂SO₄,filtered and concentrated in vacuo. The purification using flashchromatography (20% EtOAc in hexanes) yielded II24 as a waxy solid (13mg, 54% over two steps).

Characterization data for I14: δ_(H) (500 MHz, CDCl₃) 7.64 (dd, 1H, J2.5 Hz and 9.0 Hz), 8.05 (d, 1H, J 9.0 Hz), 8.14 (d, 1H, J 2.5 Hz), 8.63(d, 1H, J 2.0 Hz), 9.15 (d, 1H, J 2.0 Hz) δ_(c ()500 MHz, CDCl₃) 116.3,117.5, 120.0, 121.7, 122.8, 125.4, 130.8, 141.2, 149.1, 151.4, 151.6.

7-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)quinoline-3-carbonitrile(I15): I14 (5.7 mg, 0.0189 mmol, 1.0 eq) and I3 (13 mg, 0.0567 mmol, 3.0eq) were dissolved in DME (1 mL) at room temperature. PPh₃ (1.00 mg,0.00377 mmol, 20 mol %), KF dihydrate (5.30 mg, 0.0567 mmol, 3.0 eq),and Pd₂bda₃ (1.00 mg, 0.000943 mmol, 5 mol %) were added to thesolution. Distilled water (10 drops) was added to the resulting mixtureand the reaction was degassed and purged with argon. The reactionmixture was stirred at reflux for 16 hours. After cooling, the reactionmixture was filtered through a layer of Celite and the filtratepartitioned between EtOAc (5 mL) and H₂O (5 mL). The aqueous layer wasfurther extracted with EtOAc (5 mL). The combined organic layer waswashed with brine, dried with anhydrous Na₂SO₄, filtered andconcentrated in vacuo. The crude mixture was purified using flashchromatography (20% EtOAc in hexanes) to give I15 (4.89 mg, 76%).

Characterization data for I15: δ_(H) (300 MHz, CDCl₃) 1.34 (s, 6H), 1.37(s, 6H), 1.74 (s, 4H), 7.43 (d, 1H, J 8.1 Hz), 7.51 (dd, 1H, J 2.1 Hzand 8.1 Hz), 7.70 (d, 1H, J 2.1 Hz), 7.83 (bs, 2H), 8.02 (bs, 1H), 8.37(s, 1H), 8.80 (d, 1H, J 1.5 Hz). δ_(c) (300 MHz, CDCl₃) 31.9, 34.2,35.1, 125.5, 126.5, 144.8, 116.3, 117.5, 120.0, 121.7, 122.8, 125.4,130.8, 141.2, 149.1, 151.4, 151.6.

Methyl7-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)quinoline-3-carboxylate(I16): I15 (10.0 mg, 0.0294 mmol) was dissolved in MeOH (2 mL) to whichconcentrated HCl (2 mL) was added. The mixture was heated to reflux withstirring for 16 hours then cooled in an ice bath. The pH was adjusted toneutral with 1M KOH. The reaction mixture was extracted with EtOAc (5mL×3). The combined organic layer was washed with brine, dried withanhydrous Na₂SO₄, filtered and concentrated in vacuo. The crude mixturewas purified using preperative thin layer chromatography (30% EtOAc inhexanes) to give I16 (9.50 mg, 87%).

Characterization data for I16: δ_(H) (300 MHz, CDCl₃) 1.34 (s, 6H), 1.37(s, 6H), 1.74 (s, 4H), 4.03 (s, 3H), 7.46 (d, 1H, J 8.4 Hz), 7.53 (d,1H, J 8.1 Hz), 7.73 (s, 1H), 7.89 (d, 1H, J 8.1 Hz), 7.98 (d, 1H, J 8.4Hz), 8.39 (s, 1H), 8.87 (s, 1H), 9.46 (s, 1H).

N-hydroxy-7-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)quinoline-3-carboxamide(C): I16 (9.50 mg, 0.0254 mmol, 1.00 eq) was dissolved in dry THF (2 mL)and cooled in an ice bath. To the solution were added MeOH (0.5 mL) and50% v/v NH₂OH (in water, 0.5 mL, excess). The mixture was allowed towarm up slowly to room temperature and stirred for three days. The pHwas adjusted to approximately pH 3 with 1M KOH. The resulting solutionwas partitioned between water and EtOAc. The aqueous layer was furtherextracted with EtOAc (5 mL×3). The combined organic layer was washedwith brine, dried with anhydrous Na₂SO₄, filtered and concentrated invacuo. The crude product was purified by reverse-phase flashchromatography (20-95% MeOH in H₂O) to yield C as a clear film (6.5 mg,74%).

Characterization data for C: δ_(H) (300 MHz, CD₃OD) 1.32 (s, 6H), 1.37(s, 6H), 1.77 (s, 4H), 7.48 (d, 1H, J 8.4 Hz), 7.58 (d, 1H, J 8.1 Hz),7.75 (s, 1H), 7.94 (d, 1H, J 8.1 Hz), 7.98 (d, 1H, J 8.4 Hz), 8.39 (s,1H), 8.95 (s, 1H), 9.50 (s, 1H). δ_(c) (300 MHz, CD₃OD) 31.3, 31.4,34.1, 34.4, 35.2, 35.3, 123.5, 124.3, 124.5, 124.7, 125.20, 126.4,127.8, 128.6, 128.9, 129.7, 131.6, 136.6, 137.5, 140.7, 148.3, 149.1,167.9. HRMS (ESI) calcd for C₂₄H₂₆N₂O₂, [M−H⁺] 373.1992, found 373.1978.

Example 4 Synthesis of Compounds 22, 23, and 24

To 200 mg of 6-bromoindole 2-carboxylic acid methyl ester in THF 6 mlwas added [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)(70 mg), dicyclohexylmethylamine (1.2 ml) and boronic acid (300 mg). Themixture was stirred under reflux for 12 hours then cooled to roomtemperature. The reaction mixture was poured into water and extractedwith ethyl acetate, and the resulting organic layer was dried andevaporated. The crude mixture was purified by flash chromatography (ISCOcombiflash) using a 10-50% ethyl acetate gradient to afford 200 mg ofproduct, which was dissolved in 10 ml methanol:THF (1:1) and 2 ml of 2NKOH was added to the resulting solution. The solution was heated for 12hours at 60° C. then cooled to room temperature. Water (50 ml) and 2NHCl (4 ml) was added to the solution, which was then extracted withethyl acetate (100 ml). The organic layer was dried and evaporated. Thecrude product was purified by combiflash using 10-100% ethyl acetategradient to afford the acid as a white solid (140 mg). The acid wasdissolved in DMF (5 ml) to which EDCI (140 mg), HOBT (120 mg) and1,2-phenylenediamine (120 mg) was added and the reaction mixture wasstirred overnight at room temperature. The reaction solution wasextracted with water (100 ml) and ethyl acetate (100 ml), and theisolated organic layer was dried and evaporated. This crude product waspurified using combiflash with 0-100% ethyl acetate to give 70 mg offinal product (22).

Characterization data: (300 MHz, CD3COCD₃) 10.8 (s, 1H), 9.2 (s, 1H),7.8 (s, 1H), 7.75 (d, 1H), 7.65 (s, 1H). 7.45-7.5 (m, 5H), 7.1 (m, 1H),6.9 (m, 1H), 4.7 (bs, 2H), 1.75 (s, 4H), 1.4 (d.6H). MS: [M+1] 438.

Synthesis of Compound 23

To 250 mg of indole-5-carboxylic acid was added 16 ml of acetic acid,300 mg of boronic acid and 70 mg palladium acetate. The reaction wasflushed with oxygen, and then stirred for 48 hrs. The solvent wasevaporated, and the resulting product purified by flash chromatography(ISCO combiflash) using a 10-50% ethyl acetate/hexane gradient. Thepurified acid product was dissolved in 5 ml of DMF, and HOBt (150 mg),EDCI (150 mg), and 1,2-phenylenediamine (150 mg) was added to thesolution. The reaction was stirred overnight at room temperature. Thisreaction mixture was poured into water, extracted with ethyl acetate,dried and evaporated. A 5:1 hexane/ethyl acetate solution was added tothe crude product, and the suspension was filtered to afford 100 mg ofproduct (23).

Characterization data: NMR (600 MHz, CD3COCD₃): 11.7 (s,1H), 9.5 (s,1H),8.35 (s,1H), 7.8 (s,1H), 7.74 (d,1H), 7.66 (d,1H), 7.47 (d,1H), 7.2(d,1H), 7.01 (m,2H), 6.8 (d,1H), 6.63 (m,1H), 4.9 (s,2H), 1.66 (s,4H)and 1.3 (d,12H).

Synthesis of Compound 24

To a solution of 5-bromoindole-2-carboxylic acid methyl ester (250 mg)in THF (6 ml) was added[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (70 mg) andboronic acid (300 mg). This reaction mixture was placed degassed withargon and heated at 60° C. for 48 hours. The solvent was evaporated, andthe resulting product purified by flash chromatography (ISCO combiflash)using a 10-50% ethyl acetate/hexane gradient. The resulting product(purified ester) was hydrolyzed by heating at 60° C. overnight in asolution of methanol/THF/2N NaOH (1:1:1, 9 ml). This solution was pouredinto a solution of 5 ml 2N HCl and 50 ml water, and the extracted withethyl acetate. The organic layer was dried and evaporated. The crudeacid (100 mg) was dissolved in DMF (10 ml) to which EDCI (150 mg), HOBt(150 mg) and 1,2-phenylenediamine (150 mg) was added, and this solutionwas stirred overnight. The solvent was evaporated off, and the resultingproduct purified by flash chromatography (ISCO combiflash) using a10-50% ethyl acetate/hexane gradient to provide compound 24.

Characterization data: NMR(600 MHz, CD3SOCD3): 11.6 (s, 1H), 9.75(s,1H), 7.9 (s,1H), 7.4-7.7 (m,6H), 7.25 (9d,1H), 7.0(m,1H), 6.8 (s,1H),6.6(s,1H), 4.95 (s,2H), 1.75 (s,4H) and 1.3 (d,12H).

Example 5

The effect of compounds on the enzymatic activity of purifiedrecombinant human HDAC-1, -2, -3, -6, and -8 activity and of purifiedrat liver HDAC was examined by measuring the deacetylation of syntheticpeptides in various preparations of HDACs. HDAC assays were performedusing a two-step enzymatic reaction where enzyme activity is correlatedto the release of 4-amino-7-methylcoumarin (AMC). AMC fluorescence ismeasured in a fluorescent plate reading using λ_(ex)380 nm and λ_(m)440nm. The assay was run in a 96-well format using an assay buffer (50 mMTris-HCl, pH 8, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂ and 50 μg/mLultra-pure, non-acetylated BSA. Purified human recombinant HDAC-1,HDAC-2, HDAC-3/NCOR and HDAC-6 were assayed using Ac-Arg-Gly-Lys(Ac)-AMC (Bachem 4044046) as a substrate. Purified human recombinantHDAC-8 was assayed using Boc-Lys(Tfa)-AMC (Bachem 4060676) as asubstrate. Both substrates were used at a final concentration of 10 μM.Final enzyme concentrations in the assays were as follows: 0.38 μg/mLHDAC-2 (Cayman Chem 10009377), 1.37 μg/mL HDAC-6 (Cayman Chem 10009465),HDAC-8 (Cayman Chem 10009465, 150 μl, diluted 100×), purified rat liverHDAC (Millipore 382165, 0.8 mg/mL proteins diluted 50×).

HDAC-8, HDAC-9, HDAC-10 and HDAC-11 were assayed using HDAC-Glo™ I/IIKit (Promega Corporation G6421) in a total volume of 110 μl followingthe manufacturer's instructions. Enzyme concentrations were as follows:230 ng/μL HDAC-8 (Cayman Chem 19380), 100 ng/mL HDAC-9 (Sigma SRP5268),460 ng/ml HDAC-10 (Axxora/Enzo BML-SE99), 600 ng/mL HDAC-11 (Axxora/EnzoBML-SE560).

Benchmark compounds, including, entinostat, panabinostat, ricolinostat,romidepsin, and suberoylanilide hydroxamic acid (SAHA) were runalongside the test compounds. The compounds were dissolved in DMSO andtested in duplicate using ⅓ dilution concentration curve. Typically,HDAC preparations were pre-incubated with compounds or DMSO in assaybuffer on ice for 5 min prior to substrate addition. The reaction wasallowed to proceed for 40 min at 37° C., after which developer solution(0.5% trypsin, 10 μM trichostatin A (TSA)) was added to obtain a finalconcentration of 0.1% trypsin, 2 μM TSA. After 30 minutes at roomtemperature, the amount of free fluorogenic AMC was measured asdescribed above. Data showing the effect of various compounds of theinvention on HDAC-1 and HDAC-6 are presented in FIGS. 5A and 5B,respectively, and data for all HDACs are summarized in Table 1.

TABLE 1 1 2 3 6 8 9 10 11 Rat liver 5 +/− ++ ++ +++ +++++ +/− +/− +/− ++− 8 − − +++ ++ − − − − ++ − 13 +/− ++ ++ ++++ +++ +/− +/− +/− +++ +++ −17 − − − ++ ++ − − − − ++ − 18 +/− ++ − +++ ++ NT NT NT NT + − 19 − ++ −+++ − NT NT NT NT − − 20 − − ++ ++ NT NT NT +/− +++ 21 − − − − − − NT NT− − − Entinostat +++ ++++ +++ − + +++ ++ +++ NT − NT Panobinostat ++++++++++ +++++ +++++ +++++ +++++ +++++ +++++ NT − +++++ Ricolinostat ++++++++ +++++ +++++ ++++ NT ++ +++ NT − +++ Romidepsin +/− +/− +++ − +++++++ +++ +++ NT − ++++ SAHA ++++ ++++ +++++ +++++ +++++ +++++ ++++ +++++++++ − +++++

Because absolute IC₅₀ values vary with different assay systems orsubstrates, the relative activity of each compound is represented usingthe symbols “+” or “−”. A greater number of “+” signs denotes a greaterpotency (IC₅₀) the inhibition of the respective HDACs. The symbol “+/−”denotes that 100% inhibition of the enzyme could not be reached in thecondition used. “NT”=not tested.

Results: Under the experimental conditions, the compounds were mostlyinactive active against HDAC-1. However, compounds 5, 13, and 18inhibited up to ˜50% HDAC-1 activity. All of the compounds testeddemonstrated an apparent lack of inhibition of HDAC-1, -2, -3, -9, -10and -11 under the experimental conditions. Surprisingly, compound 5 wasshown to be over 200 times more potent than compound 17 in the HDAC-8inhibition assay. Likewise, compound 13 was shown to be at least 50-foldmore potent than compound 17 in the HDAC-6 inhibition assay (FIG. 5A andTable 1), and at least 13-fold more potent than compound 17 in HDAC-8inhibition assay (Table 1).

When measuring inhibition of purified rat hepatic HDAC, compound 8 wasat least three times more potent than compound 17, and compounds 5 and13 were six and seventy times more potent than compound 17, respectively(Table 1).

Example 6

The effect of various compounds of the invention on RAR-mediatedtranscription in MCF7 breast cancer cells was examined by measuringtheir effect on the transcription of a luciferase reporter gene underthe control of a TK promoter placed downstream of a RAR responseelement. In addition, the effect of the compounds on the closely relatedRXR-mediated transcription was examined by measuring their effect on thetranscription of a luciferase reporter gene under the control of a TKpromoter placed downstream of a RXR response element.

Typically, 10,000 cells were plated in 96 wells plates in phenolred-free RPMI medium supplemented with 10% charcoal-stripped FetalBovine Serum. After 24 hours, cells were transfected with 0.1 mg ofluciferase reporter plasmid and 0.01 μg of a reporter plasmid expressingrenilla luciferase (RLuc) gene under the control of a SV40 promoter,using Fugene 6 transfection reagent (0.5 Owed Promega Corporation).Cells were treated in triplicate with compounds or vehicle (DMSO) for 24hours. Luciferase and renilla luciferase activities were measured usingDual-Glo reagent (Promega Corporation). Luciferase activity wasnormalized in each well for renilla activity.

Results: As shown in FIG. 6A and summarized in Table 2, compounds 8, 13,18, 19 and 20 activated RARE-dependent luciferase expression in MCF7cells. Compound 13 was five to ten times more potent than compound 17 inactivating a RARE-tk_luc reporter plasmid transfected in MCF7 cells.Surprisingly, while all benchmark HDAC inhibitors activatedtranscription from an RXRE-promoter, none of compounds 5, 8, 13, 17, 18,19, 20 and 21 demonstrated transcriptional activation of RXR (FIG. 6B;Table 2).

TABLE 2 Summary of compounds' ability to activate RARE-dependenttranscription in MCF7 cells Compound RAR RXR  5 −  8 ++ − 13 +++ − 17 ++− 18 + − 19 − − 20 +++ 21 − − Entinostat − NT Panobinostat − +++++Ricolinostat − +++ Romidepsin − ++++ SAHA − +++++

Example 7

The cytotoxicity of compounds was examined and directly compared tocompound 17, by measuring cell survival of clonal tumor cell linesIMR-32, BE2C (neuroblastoma), as well as MDA-MB-231, BT20, HCC-38(Basal) and MCF7 (luminal) breast cancer cells (ATCC). Cells werecultured in phenol red-free RPMI medium supplemented with 10% FBS and 1mM Ala-Glu. Typically, 10,000-20,000 cells/well were plated in a 96 wellplate (Costar 3197). Cells were exposed to compounds or vehicle (1% DMSOfinal). Cells were maintained in the presence of compound for four orfive days. Media and compounds were renewed every 48 hours. Cellsurvival was measured using the luminescence CellTiterGlo kit (PromegaCorporation) and expressed as relative light unit as fold over DMSO.

Results: The data show that compounds were cytotoxic in a variety ofcancer cell lines (FIGS. 7A-C). In IMR-32 neuroblastoma cells, thecompounds tested were cytotoxic in the 30 nM to 300 nM range.Strikingly, compounds 5 and 13 were ten-fold more potent than compounds17 and 8 in killing IMR-32 cells (FIG. 7A). Strikingly, in both BE-2Cand MDA-MB-231 cancer cell lines, compounds 13 and 18 were more potentthan compound 17, SAHA or Ricolinistat (FIGS. 7B-7C).

Example 8

The effect of certain compounds on intracellular acetylation of targetproteins was tested in high risk neuroblastoma cells, BE-2C and IMR-32cells.

BE-2C cells were grown to confluence in phenol-red-free RPMI mediasupplemented with 10% FBS and 2.5 mM Ala-Glu. The cells were treatedwith DMSO or 5 μM compound 13, Ricolinostat (2 uM), SAHA (2 uM), or TSA(1 uM), in DMSO for 16 hours. Total and Lys 40 acetylated alpha tubulinwere detected using specific antibodies.

IMR32 cells were grown to confluence in phenol-red-free RPMI mediasupplemented with 10% es grade FBS and 2.5 mM Ala-Glu. Cells weretreated with DMSO or 1 μM or 5 μM compound 13 in DMSO for 16 hours.Protein extracts were prepared in RIPA buffer in the presence ofprotease and phosphatase inhibitors. Proteins were separated on a 4-20%gel by PAGE and transferred to a nitrocellulose membrane.

Beta actin, total and Lys382 acetylated p53, and Lys40 acetylated alpha,and total tubulin were detected using antibodies (Cell Signaling #8557,#2524 #2525, #12152 and #2144 respectively) using manufacturerrecommended conditions.

Results: The data show that, consistent with the HDAC inhibitoryactivity observed in vitro (Table 1), under conditions where neither thelevel of total p53 nor that of total beta actin were affected, additionof 1 μM or 5 μM compound 13 resulted in a pronounced accumulation oflysine acetylated p53 (FIG. 8). Surprisingly, while treatment withricolinostat, SAHA and TSA all resulted in a pronounced Lysin (K) 40alpha-Tubulin acetylation, no significant acetylation was observed incompound 13-treated cells at 5 μM, a concentration greater than IC₅₀(FIG. 9). The lack of increased alpha tubulin acetylation was confirmedusing LC-MS analysis. These findings confirm that compound 13 acts as abona fide inhibitor of HDAC activity, and suggests that the cytotoxicityof this compound does not result from tubulin acetylation.

Example 9

The effect of compounds on myoblast fusion was examined in C2Cl2 cellsmaintained in RPMI media supplemented with 2% horse serum. C2Cl2myoblastic cells where purchased from the American Type CultureCollection (ATCC) and maintained undifferentiated in RPMI media(Corning) supplemented with 2 mM L-Alanine-L-Glutamine (ATCC) and 1 mMpyruvate (Corning). Undifferentiated cells where maintained below 70%confluence. For long term cultures and compound treatment, cells whereplated at high density CellBIND Culture Dishes (corning 3294, 3296,3337) and in RPMI supplemented with 2% Horse Serum (Gibco) and 2 mML-Alanine-L-Glutamine (ATCC) and 1 mM pyruvate (Corning). Compounds weredissolved in DMSO at 1000 time the final concentration and diluted1-in-1000 in the media covering the cells. Media and compounds werechanged daily. For immunocytochemistry, cells were fixed in 2% PFA inHBSS for 10 minutes and left in HBSS at 4° under humidified atmosphereand processed for immunocytochemistry. For myosin heavy chain detection(MHC), cells were first incubated in 100 ul-1 ml 0.15 M Glycine in DPBSfor 10 minutes, rinse with DPBS. 100 ul-1 ml 0.5% Triton-X in DPBS wasadded for 10 minutes, rinsed with DPBS, and followed by incubation with100 ul blocking agent (10% goat serum in DPBS) for 30 min at roomtemperature. MF20 primary antibody in 10% goat serum in DPBS was addedat 1/60 dilution for 30 min at room temperature and rinsed three timeswith 100 ul DPBS. IgG2b goat anti mouse secondary antibody (JacksonImmuno-Research) was added at 1:1000 in 10% goat serum in DPBS forexactly 30 min at room temperature then rinsed 3 times with 100 ul DPBS.A final rinse in H₂O was performed and cells were covered with two dropsof Elvanol and cover gently with cover slip and allowed to dry in thedark for 24-48 hours.

Results: Surprisingly, our data suggest that under long-term cultures insuboptimal RPMI-containing media, addition of compounds 13, 17 or Benhances the fusion of single cells into large multinucleated, elongatedC2Cl2 myotubes (FIG. 10) Immunohistochemistry staining assays suggestthat the compounds promote the fusion and/or maintenance of myosin heavychain (MHC)-expressing differentiated myoblasts. In DMSO-treated C2Cl2cells cultured for 10 days, myotube formation does not occurs despitethe presence of a large number of almost exclusively mononucleatedMHC-expressing differentiated myoblasts with a singlepolynucleated-MHC-expressing myotube. Under the same conditions, using300 nM of compound 17 resulted in the majority of MHC-expressing cellsbeing polynucleated myotubes with an average of 5-10 nuclei per cell,suggesting that fusion was promoted (FIG. 11).

The quantitation of the average distribution of myotubes' length anddiameter demonstrate that in the presence of 300 nM of compound 17 arelonger and larger than when treated with DMSO (FIGS. 12A and 12B).

Taken together, our data suggest that the compounds tested significantlyimprove myoblast fusion and/or myotube survival in long-term cultures.

Example 10

C2Cl2 myoblasts were seeded at 100,000 in 30 mm plate in growth medium(Phenol red-free RMPI containing 10% FBS, Sodium pyruvate and Ala-Glu),and “pre-treated” with DMSO or 200 nM of compounds 13 or 17. Atconfluence cells were shifted to differentiation media (high glucoseDMEM, +0.2% HS+1× Insulin/Transferrin/Selenium (Life Tech41400-045)+Glutamine, without Pyruvate). Fully differentiatedC2Cl2-derived myotubes were left in culture without media renewal for 8days followed by media renewal and microscopic examination.

Results: The data show that extensive cellular fusion was observed after72 hours differentiation in cells that were treated with compounds 13 or17 versus DMSO (FIG. 13 top).

In addition, when left in exhausted media for over 8 days, DMSO-treatedmyotubes, but not myotubes treated with compounds 13 or 17, underwentsevere structural atrophy and cell death. In contrast, myotubes treatedwith compounds 13 or 17 withstood culture stress conditions with verylittle structural alteration (FIG. 13 bottom).

Example 11

Treatment with compounds 13 or 17 down regulates the expressionmuscle-atrophy genes myogenin and Atrogin in long term C2Cl2 cultures(FIGS. 14A and 14B).

Quantitative RT-PCR was performed on RNA prepared from cell treated asin Example 9. At day 10 of the experiment, cells were rinsed in HBSS(without calcium or magnesium) and RNA was prepared using FastLane CellcDNA Kit (Qiagen). Quantitative PCR was performed using Eppendorf andSYBR amplification mix (Sigma-Aldrich) using the gene specific primercombination given in Table 3. Data were normalized for ribosomalhousekeeping gene RPLP0

Results: Consistent with a role in preserving myotube integrity duringlong term cultures, compounds 13 and 17 down-regulated the expression ofgenes implicated in myofiber degradation.

TABLE 3Sequence of gene-specific primers used in Q-RT PCR quantitation of myofiber-degradation associated genes. ATROGIN MYOGENIN RPLP0 Forward PrimerCTTCTCAGAGAGGCAGATTC CCCAACCCAGGAGATCAT CGGAGGAATCAGATGAGGATA(SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ ID NO: 5) Reverse PrimerTCTTCTTGGGTAACATCGTACA CTGGGAAGGCAACAGACATA CAGACCGGAGTTTTAAGAGAAG(SEQ ID NO: 6) (SEQ ID NO: 1) (SEQ ID NO: 2)

Example 11

Mouse Pharmacokinetics studies were conducted in female athymic nude ormale C57/B6 mice (n=3). Due to its very poor aqueous solubility,compound 17 was formulated in 66% PEG-400/33% H₂O-1% Tween-80. Incontrast compound 13 was formulated in 66% PEG-400/33% H2O. Compoundswere administered PO (30 mg/kg) or IV (5 mg/kg), and plasmaconcentrations were measured by UV/HPLC. The results are presented inFIG. 15 and pharmacokinetic parameters for each compounds are summarizedin Table 4.

TABLE 4 Compound 17 Compound 13 AUC_(0-∞) (μM * h) 28.71 42.00 C_(max)(μM) 2.08 5.54 T_(max) (h) 4.3 1.4 F (%) 19.7 ~40 CLp (mL/min/kg) 10.20.24 Vd, ss (L/kg) 4.6 8 MRT (h) 7.1 6.9

Results: Compound 13 displayed improved aqueous solubility andpharmacokinetics properties compared with compound 17. Notably, oralbioavailability, AUC, Cmax were markedly higher for compound 13, while Tmax was markedly reduced (FIG. 15 and Table 4).

What is claimed is:
 1. A compound of Formula I: A-W—Z (I) or apharmaceutically acceptable salt thereof, wherein A is

W is a heterocyclylene, arylene, heteroarylene, alkenylenearylene,arylenealkenylene alkenyleneheteroarylene, or heteroarylenealkenylene;and Z is —C(O)NR¹R², wherein R¹ and R² are each independently hydrogen(H), hydroxyl (OH), C₁₋₆ alkyl, hydroxyC₁₋₆ alkyl, aminoC₁₋₆ alkyl, oraminoaryl; with the proviso that the compound is not


2. The compound of claim 1, wherein W is an indolinylene linked to A atany one of positions 2, 3, 4, 5, 6 or 7 of the indolinylene; aquinolinene linked to A at any one of positions 2, 3, 4, 5, 6, 7, or 8;or an isoquinolinene linked to A at any one of positions 1, 3, 4, 5, 6,7, or
 8. 3. The compound of claim 1, wherein W is -propylene-phenylene-.4. The compound of claim 2, wherein Z is linked to the indolinylene,quinolinene, or isoquinolinene at any one of the positions that is notlinked to A.
 5. The compound of claim 1, wherein W is


6. The compound of claim 1, wherein W is an indolinylene linked to A atany one of positions 2, 3, 4, 5, 6 or 7 of the indolinylene; Z is—C(O)NR¹R², wherein R¹ and R² are each independently hydrogen, hydroxyl,C₁₋₆ alkyl, hydroxyC₁₋₆ alkyl, aminoC₁₋₆ alkyl, or aminoaryl; andwherein Z is linked to the indolinylene at any one of positions 2, 3, 4,5, 6 or 7 of the indolinylene not linked to A.
 7. The compound of claim1, wherein W is a quinolinene linked to A at any one of positions 2, 3,4, 5, 6, 7 or 8 of the quinolinene; Z is —C(O)NR¹R², wherein R¹ and R²are each independently hydrogen, hydroxyl, C₁₋₆ alkyl, hydroxyC₁₋₆alkyl, aminoC₁₋₆ alkyl, or aminoaryl; and wherein Z is linked to thequinolinene at any one of positions 2, 3, 4, 5, 6, 7 or 8 of thequinolinene.
 8. The compound of claim 1, W is a isoquinolinene linked toA at one of positions 1, 3, 4, 5, 6, 7 or 8 of the isoquinolinenemoiety; Z is —C(O)NR¹R², wherein R¹ and R² are each independentlyhydrogen, hydroxyl, C₁₋₆ alkyl, hydroxyC₁₋₆ alkyl, aminoC₁₋₆ alkyl, oraminoaryl; and wherein Z is linked to the quinoline ring at any one ofpositions 2, 3, 4, 5, 6, 7 or 8 of the isoquinolinene.
 9. The compoundof claim 6, wherein Z is —C(O)NR¹R²; R¹ is H; and R² is OH or aminoaryl.10. The compound of claim 7, wherein Z is —C(O)NR¹R²; R¹ is H; and R² isOH or aminoaryl.
 11. The compound of claim 8, wherein Z is —C(O)NR¹R²;R¹ is H; and R² is OH or aminoaryl.
 12. The compound of claim 1, whereinthe compound is


13. A pharmaceutical composition comprising a compound of claim 1, and apharmaceutically acceptable carrier.